Figure 6: Effect of micropillar confinement on the orientation of the mitotic spindle.

To assess the angle and height of the mitotic spindle, H2B-mCherry (red)- and tubulin-eGFP (green)-expressing HeLa cells were cultured on DiD-labelled micropillar arrays (blue) and cell culture plates. Micropillars had average distances of ≈6.8 μm and lengths of ≈8.2 μm. Confocal images were taken of HeLa cells in metaphase in (a) absence or (b) presence of 10 μM Y-27632. Lateral scale bars, 3.5 μm and vertical scale bars, 5 μm. (c) Determination of the spindle angle and height of a confined mitotic cell (i) showed that unperturbed HeLa cells orient their spindle parallel (towards an angle θ of 0°) to the substrate plane, (ii) whereas cells cultured in the presence of 25 μM Y-27632 possessed a more vertical orientation (towards an angle θ of 90°) of the mitotic spindle (ii). The angle (d) and the height (e) of the mitotic spindle were influenced by chemical compounds perturbing the actomyosin cortex. The confining micropillars enhanced the perturbation of the mitotic spindle angle and height. Each dot represents one experimentally characterized cell. For each condition, the bar denotes the average. Mann–Whitney P values indicate the significance relative to the control (untreated) cells. NS, not significant, P≥0.05, *P<0.05, **P<0.01, ***P<0.001. Perturbants are described in Supplementary Table 1.